A broad-host-range mobilizable shuttle vector for the construction of transcriptional fusions to β-galactosidase in Gram-positive bacteria

FEMS Microbiology Letters 156 (1997) 193^198

A broad-host-range mobilizable shuttle vector for the construction of transcriptional fusions to L-galactosidase in Gram-positive bacteria Claire Poyart, Patrick Trieu-Cuot * è INSERM 411, Faculte è de Me èdecine Necker-Enfants Malades, 75730 Paris Cedex 15, France Laboratoire de Microbiologie, Unite

Received 21 August 1997; revised 10 September 1997; accepted 12 September 1997

Abstract

A low-copy-number vector designated pTCV-lac has been constructed to provide a convenient system to analyze regulatory elements in Gram-positive bacteria. The main components of this vector are: (i) the origins of replication of pACYC184 and of the broad-host-range enterococcal plasmid pAML1, (ii) erythromycin- and kanamycin-resistance-encoding genes for selection in Gram-negative and Gram-positive bacteria, (iii) the transfer origin of the IncP plasmid RK2, and (iv) a promoterless Lgalactosidase-encoding lacZ gene with a Gram-positive ribosome binding site. This 12 kb plasmid is present in Gram-positive hosts in three to five copies per chromosome equivalent and contains three unique cloning sites (EcoRI, SmaI, BamHI) for cloning of DNA inserts upstream of the lacZ gene. Plasmid pTCV-lac and derivatives carrying different promoter fragments have been transferred by conjugation from an Escherichia coli IncP mobilizing donor strain to Bacillus subtilis, Listeria monocytogenes, Enterococcus faecalis, and Streptococcus agalactiae. These plasmids were structurally stable in these hosts and the corresponding promoter activities, quantitated by the determination of the L-galactosidase specific activities, were found to cover at least a 100-fold range in L-galactosidase values. These results indicate that pTCV-lac should be useful for analysis of gene regulation in a wide range of Gram-positive bacteria. Keywords :

Mobilizable shuttle vector; Transcriptional fusion; L-galactosidase; Gram-positive bacteria

1. Introduction

The most convenient and versatile systems developed to study the activity of promoters in vivo usually consist of a plasmid-borne promoterless gene that codes for easily assayable products such as the L-galactosidase [1] or chloramphenicol acetyltransferase [2]. DNA fragments to be analyzed for pro* Corresponding author. Tel.: +33 (1) 40 61 56 79; Fax: +33 (1) 40 61 55 92; E-mail: [email protected]

moter activity can then be inserted upstream from the reporter gene and measurements of the amount of gene product are used to determine the level of transcription from the promoter(s) within the fragment. Numerous plasmid vectors enabling transcriptional fusions to either promoterless L-galactosidase or chloramphenicol acetyltransferase coding genes have been constructed for the analysis of gene control elements in Gram-positive bacteria [3^6]. The major disadvantage of the existing plasmid systems developed to date to measure promoter activity in

0378-1097 / 97 / $17.00 ß 1997 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PII S 0 3 7 8 - 1 0 9 7 ( 9 7 ) 0 0 4 2 3 - 0

FEMSLE 7851 10-11-97

194

C. Poyart, P. Trieu-Cuot / FEMS Microbiology Letters 156 (1997) 193^198

Gram-positive bacteria is their high copy number per cell which precludes their utilization for studying the ¢ne regulation of promoter activities in vivo. Moreover, they often display a low versatility as a consequence of their narrow host range of replication. We describe here the construction and use of an IncP-based mobilizable shuttle vector that enable transcriptional fusion to L-galactosidase in a wide range of Gram-positive bacteria (Bacillus, Enterococcus, Listeria, Streptococcus) where it replicates at a low copy number per cell. 2. Materials and methods

2.1. Bacterial strains, growth conditions and genetic techniques E. coli TG1 [7] and SM10 [8] were used as hosts for plasmid construction and plasmid transfer, respectively. The Gram-positive strains used in this study (Bacillus subtilis NEM134, Listeria monocytogenes L028, Enterococcus faecalis BM4110 and Streptococcus agalactiae NEM316) were from our laboratory collection. All microorganisms were grown in brain heart infusion broth or agar at 37³C. Recombinant plasmid DNA were introduced by transformation into E. coli [9]. Filter matings between E. coli SM10 and Gram-positive recipients were carried out as described [10]. After 18 h of incubation at 37³C, the cells were suspended in 2 ml of broth and plated on agar containing appropriate antibiotics to select transconjugants. Transfer frequencies are expressed as the number of transconjugants per donor colony forming unit (cfu) after the mating period. 2.2. DNA manipulations

Plasmid DNA were extracted from E. coli as described [9]. DNA fragments of 225 bp containing the tac (Ptac) or the trc promoter (Ptrc) were ampli¢ed by PCR from plasmid pKK233-2 or pTcr99A [11], respectively, by using the oligonucleotides (oligos) TcrO1 (5P-CGGAATTCTGGCGTCAGGCAGCCATC-3P) and TcrO2 (5P-CGGGATCCTGTGTGAAATTGTTATCC-3P). A 307 bp DNA fragment containing the spac promoter (Pspac) was ampli¢ed

by PCR from plasmid pDH88 [12] by using the oligos SpacO1 (5P-CGGAATTCTACACAGCCCAGTCCAGAC-3P) and SpacO2 (5P-CGGGATCCCCGGGAAAAGCTTAATTG-3P). A 369 bp DNA fragment containing the aphA-3 promoter (PaphA3) [13] was ampli¢ed by PCR from plasmid pAT21 [14] by using the oligos KanO1 (5P-CGGAATTCCCAGCGAACCATTTGAGG-3P) and KanO2 (5PCGGGATCCGATTTTGAAACCAC-3P). All oligos were designed to add EcoRI and BamHI sites (underlined bases) upstream and downstream from the ampli¢ed promoters, respectively. The ampli¢ed fragments were digested with EcoRI plus BamHI and cloned into pTCV-lac. The fragments inserted into pTCV-lac were sequenced to verify that no misincorporation of nucleotides occurred during the PCR assay. This was done by using the oligos Vlac1 (5PGTTGAATAACACTTATTCCTATC-3P) and Vlac2 (5P-CTTCCACAGTAGTTCACCACC-3P), the dye terminator method, and an ABI-Prism 310 automatic sequencer (Perkin Elmer, Applied Biosystem Division, Roissy, France). 2.3. L-galactosidase assays

Cells were grown overnight in BHI broth containing kanamycin (B. subtilis and L. monocytogenes, 50 Wg/ml; E. faecalis and S. agalactiae, 1000 Wg/ml). Overnight cultures were diluted 50-fold in BHI broth supplemented with kanamycin and cells were collected in early stationary phase. L-galactosidase was assayed as described [15] except that the cells were permeabilized by treatment with 0.5% toluene and 4.5% ethanol. The L-galactosidase speci¢c activities, determined in three experiments, were expressed as (103 U(OD420 of the reaction mixture31.75 OD550 of the reaction mixture)) divided by (time of the reaction (min)UOD600 of the quantity of cells used in the assay). 3. Results and discussion

3.1. Construction and functional properties of plasmid pTCV-lac

The mobilizable shuttle vector pAT187 contains the origin of replication of pBR322 and that of the

FEMSLE 7851 10-11-97

C. Poyart, P. Trieu-Cuot / FEMS Microbiology Letters 156 (1997) 193^198

cus)

195

[10] where it exhibits a high segregational and

structural stability. To construct pTCV-lac, the 2.2

AvaI

kb

oriR of PvuII-SspI

fragment containing the pBR322

pAT187-1 was replaced by the 904 pb

oriR

fragment containing

of plasmid pACYC184

[17]. In a subsequent step, the 752 pb

EcoRI-PstI

fragment of the resulting vector was replaced by the

3.85

kb

EcoRI-PstI

pMC11 [5] containing the

fragment

ermB

of

plasmid

gene of the enter-

ococcal transposon Tn917 [18] and the promoterless

spoVG-lacZ

translational gene fusion [19] (Fig. 1).

Plasmid pTCV-lac has a size of 12 kb, contains three unique restriction sites (EcoRI, immediately

(i) a 3.85 kb PstI-EcoRI fragment containing the ermB gene of the enterococcal transposon Tn917 [18] and the spoVG-lacZ translational gene fusion [19] ; (ii) a 0.6 kb AvaI-PstI fragment containing the transfer origin oriT of RK2 [21] ; (iii) a 0.9 kb SspI-PvuII fragment containing oriR of plasmid pACYC184 [17] ; (iv) a 1.65 kb EcoRI-AvaI containing the kanamycin resistance gene aphA-3 from the enterococcal plasmid pJH1 [14] ; and (v) a 5 kb EcoRI fragment containing oriR of the en-

(Fig.

1),

terococcal plasmid pAM 1 [16]. Heavy arrows indicate the direc-

tain the main functional properties of the parental

tion of transcription of the genes. Three unique cloning sites

plasmid pAT187 : (i) it replicates at a low copy num-

Fig. 1. Structure of plasmid pTCV-lac. The components of this vector are :

SmaI, BamHI) lacZ gene. The

enable cloning of DNA inserts upstream

of the

positions of the oligos Vlac1 and Vlac2

150

50

form

pTCV-lac.

Only

relevant

restriction

shown. Nucleotide sequence numbering begins at the

sites

EcoRI

and 50

resistance

to

erythromycin

E. coli (selective concentrations,

Wg/ml,

respectively), in Gram-pos-

Wg/ml,

Wg/ml,

Wg/ml

and

respectively), and in Gram-positive anae-

robes (selective concentrations, 10

Wg/ml

and 1000

respectively). This vector was designed to re-

ber in Gram-positive hosts which enable an accurate characterization of promoter activities in vivo, (ii) it contains the transfer origin of the IncP plasmid RK2

tion sites in parentheses were destroyed by DNA polymerase and to

Wg/ml

from

itive aerobes (selective concentrations, 10

used to sequence the cloned fragments are indicated. The restric-

fused

confers

and to kanamycin in

L

(EcoRI,

and

upstream

SmaI, BamHI) lothe lacZ gene

cated

which enables its transfer by mobilization from an

are

coli

site.

E.

mobilizing donor to various Gram-positive ba-

cilli and cocci.

L

3.2. Conjugal transfer of pTCV-lac and derivatives from E. coli to various Gram-positive bacteria

enterococcal broad-host-range plasmid pAM 1 [10]. This plasmid therefore replicates at low copy number, approximately 5 copies per cell [16], in a wide range of Gram-positive genera (Bacillus,

Clostridia, Listeria, Enterococcus, Staphylococcus, Streptococ-

EcoRI-BamHI

fragments containing Ptac, Ptcr,

Pspac, and PaphA-3 were inserted into pTCV-lac lin-

Table 1 Transferability of plasmid pTCV-lac from Recipient

B. L. E. S. a

subtilis NEM314 monocytogenes LO28 faecalis BM4110 agalactiae NEM316

Escherichia coli

SM10 to various Gam-positive bacteria

W

Antibiotic selection ( g/ml)

b

Transfer frequency

U 3 3 U U 33 U

Sm, 500 ; Km, 50

3( þ 4)

10

Nal, 50 ; Km, 50

3.5( þ 2.1)

Nal, 50 ; Km, 1000

7( þ 3.2)

Nal, 50 ; Km, 1000

1.8( þ 3.6)

c

(mean þ S.D.)

7

10 7

6

10

10

8

NEM134 is a streptomycin resistant mutant of Marburg W168 ; LO28, BM4110, and NEM316 are naturally resistant to nalidixic acid.

b c

a

Streptomycin (Sm) plus kanamycin (Km) or nalidixic acid (Nal) plus kanamycin were used to select transconjugants harboring pTCV-lac.

Experiments were performed in triplicate and the mean values þ standard deviations (S.D.) are indicated. Transfer frequencies are expressed

as the number of transconjugants per donor cfu after the mating period. Derivatives of pTCV-lac carrying all four promoter regions used in this study were transferred at similar frequencies.

FEMSLE 7851 10-11-97

C. Poyart, P. Trieu-Cuot / FEMS Microbiology Letters 156 (1997) 193^198

196

L-galacto-

earized with the same enzymes to direct

sidase synthesis. The transferability of pTCV-lac and

E. coli SM10 to B. subtilis, L. monocytogenes, E. faecalis, and S. agalactiae was tested by ¢lter matings. In these experiments, pTCV-lac transferred at frequencies rang-

derivatives carrying these inserts from

U

36

U

38 ,

3

10 sequence) promoters. Ptac displays a spac-

and

3

3

35 (TTGACA)

10 (TATAAT) sequences compared to a spac-

trc

ing of 17 bp between these regions in the

pro-

moter [11]. Ptac was found to mediate low levels of

L-galactosidase

synthesis in

B. subtilis

which con-

¢rmed [20] that it was weakly active in this bacterial

the recipient strain considered (Table 1). Presence

species (Table 2). However, the e¤ciency of the

10

10

(

ing of 16 bp between the canonical

depending upon

ing from 3.5

to 1.8

lac

of an insert carrying promoter activity did not modify the transfer e¤ciency of pTCV-lac (data not

structurally related promoter Ptrc in

B. subtilis

was

approx. 6-fold that of Ptac which re£ects the pres-

3

shown). In every mating, restriction endonuclease

ence of the preferred 17 bp spacing between its

analysis of the plasmid content of randomly selected

and

transconjugants harbouring pTCV-lac and deriva-

associated with Ptac or Ptrc was detected in

tives carrying Ptac, Ptrc, Pspac, and PaphA-3 re-

vealed that no detectable rearrangement occurred during

the

conjugal

transfer

process

(data

not

3

10 sequences [20]. No

monocytogenes, E. faecalis,

S. agalactiae

and

35

activity

L.

(Table

2). Pspac is also a hybrid promoter constructed in

shown). Moreover, in every species, sequence analy-

vitro by joining the

sis of the plasmid content of one clone representative

B. subtilis

of each pTCV-lac derivative revealed that no micro-

L-galactosidase

3

35 region (TTGACT) of the

3

phage promoter SPO-1 and

(CATAAT) of the

E. coli lac

10 region

promoter, these two

rearrangements occurred in either of the four pro-

hexanucleotides being separated by 17 bp [12]. Pspac

moter regions studied. Taken together these results

was reported to be a strong promoter in

indicate that mobilization from an

E. coli donor con-

B. subtilis

and, accordingly, its presence in pTCV-lac was asso-

L-galactosidase

stitutes a simple and convenient mean for introduc-

ciated with a high level of

ing pTCV-lac derivatives carrying promoters into a

this bacteria (Table 2). Surprisingly, the activity of

wide range of Gram-positive hosts.

this

3.3. L-galactosidase expression from pTCV-lac in Gram-positive bacteria

resistance gene carried by the enterococcal plasmid

promoter was barely detectable in L. monocytogenes, E. faecalis, and S. agalactiae. PaphA-3 directs transcription of the kanamycin pJH1 and is constituted of a canonical

Presence of pTCV-lac in

B. subtilis, L. monocytogenes, E. faecalis, and S. agalactiae did not result

(TTGACA) separated by 17 bp from a

in the synthesis of a detectable amount of

moters characterized in this work in

L-galacto-

monocytogenes and E. faecalis where it

constructed from the

15.4-, and 12.8-fold more

3

(

35 sequence) and

3 3

35 sequence 10 sequence

(TATCTT) [13]. It is the strongest of the three pro-

sidase (Table 2). Ptac and Ptrc are hybrid promoters

E. coli trp

activity in

B. subtilis, L. mediates 1.2-,

L-galactosidase

synthesis

Table 2 Promoter activities in various Gram-positive bacteria Promoter fused to

lacZ

L-galactosidase B. subtilis

6

none Ptac

NEM314

1

7.45 þ 0.4

Ptrc

43.7 þ 2.8

Pspac

193.7 þ 7.6

PaphA-3

234.4 þ 11

a

activities (mean arbitrary units þ S.D.)

L. monocytogenes

6 6 6

LO28

1 1 1

a

E. faecalis

6 6 6

1 1 1

4110

S. agalactiae

6 6 6

NEM316

1 1 1

3.6 þ 0.5

6.2 þ 0.4

4.3 þ 0.6

55.5 þ 3.1

79.3 þ 6.3

10.5 þ 0.9

L

Experiments were performed in triplicate and the mean values þ standard deviations (S.D.) are indicated. The -galactosidase speci¢c 3 activities are expressed as (10 (OD420 of the reaction mixture 1.75 OD550 of the reaction mixture)) divided by (time of the reaction

(min)

U

U

3

OD600 of the quantity of cells used in the assay).

FEMSLE 7851 10-11-97

C. Poyart, P. Trieu-Cuot / FEMS Microbiology Letters 156 (1997) 193^198 than Pspac, respectively (Table 2). In

S. agalactiae,

its e¤ciency is 2.5-fold that of Pspac. These results are consistent with the fact that the

aphA-3

gene

associated with its natural promoter has been successfully

used

as

a

selective

marker

in

various

gene expression in

197

Escherichia coli

and yeast. Methods En-

zymol. 100, 293^308. [2] Brosius, J. (1984) Plasmid vectors for the selection of promoters. Gene 27, 151^160. [3] Fouet, A., Sirard, J.C. and Mock, M. (1994)

Bacillus anthracis

pXO1 virulence plasmid encodes a type 1 DNA topoisomerase. Mol. Microbiol. 11, 471^479.

Gram-positive bacilli and cocci.

[4] Fouet, A. and Sonenshein, A.L. (1990) A target for carbon source-dependent negative regulation of the

Bacillus subtilis. 4. Conclusions

citB

promoter of

J. Bacteriol. 172, 835^844.

[5] Debarbouille, M., Arnaud, M., Fouet, A., Klier, A. and Ra-

sacT gene regulating the sacPA operon Bacillus subtilis shares strong homology with transcriptional

poport, G. (1990) The

The objective of this work was the construction of a low-copy-number promoter-lac fusion vector for the study of gene expression in Gram-positive bacteria. The vector constructed, designated pTCV-lac, was successfully used to compare the activity of four promoters (Ptac, Ptrc, Pspac, and PaphA-3) in two

in

antiterminators. J. Bacteriol. 172, 3966^3973. [6] Band, L., Yansura, D.G. and Henner, D.J. (1983) Construc-

Bacillus subtilis.

tion of a vector for cloning promoters in Gene 26, 313^315.

[7] Gibson, T.J. (1984) Studies on the Epstein-Barr virus genome. PhD thesis. Cambridge University, Cambridge, England.

Gram-positive bacilli (B. subtilis and L. monocytogenes) and two Gram-positive cocci (E. faecalis and S. agalactiae). We have shown that the activities of

[8] Simon, R., Priefer, U. and Puhler, A. (1983) A broad host

these promoters may vary largely depending upon

[9] Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular

the host species considered. The most striking result

Cloning : A Laboratory Manual. Cold Spring Harbor Labo-

was the demonstration that Ptrc and Pspac, which function in moters,

B. subtilis

respectively,

(Pspac) active in

S. agalactiae.

as moderate and strong prowere

(Ptrc)

not

or

barely

L. monocytogenes, E. faecalis,

and

These experiments are representative

for other applications and show the usefulness and versatility of pTCV-lac. In particular, beside gene regulation studies, this vector can be used to choose the

most

appropriate

promoter

to

produce

pro-

teins in the desired amount, or only under peculiar growth conditions, in a wide range of Gram-positive hosts.

range mobilization system for in vivo genetic engineering : Transposon mutagenesis in Gram-negative bacteria. Biotechnology 1, 784^794.

ratory Press, Cold Spring Harbor, NY. [10] Trieu-Cuot, P., Carlier, C., Martin, P. and Courvalin, P. (1987) Plasmid transfer by conjugation from

Escherichia coli

to Gram-positive bacteria. FEMS Microbiol. Lett. 48, 289^ 294. [11] Amann, E., Ochs, B. and Abel, K.J. (1988) Tightly regulated

tac

promoter vectors useful for the expression of unfused and

fused proteins in

Escherichia coli.

Gene 69, 301^315.

Escherichia coli lac repressor and operator to control gene expression in Bacillus subtilis. Proc. Natl. Acad. Sci. USA 81, 439^

[12] Yansura, D.G. and Henner, D.J. (1984) Use of the

443. [13] Trieu-Cuot, P., Klier, A. and Courvalin, P. (1985) DNA sequences specifying the transcription of the streptococcal kanamycin resistance gene in

subtilis.

Escherichia coli

and in

Bacillus

Mol. Gen. Genet. 198, 348^352.

[14] Trieu-Cuot, P. and Courvalin, P. (1983) Nucleotide sequence of the

Acknowledgments

Streptococcus faecalis

plasmid gene encoding the 3P5Q-

aminoglycoside phosphotransferase type III. Gene 23, 331^

We thank S. Na|ër for critical reading of the manuscript and P. Berche for his interest in this work and

341. [15] Miller, J.H. (1972) Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

material support. This work was supported by the

[16] Swing¢eld, T.-J., Oultram, J.D., Thompson, D.E., Brehm,

è et de la Recherche Institut National de la Sante

J.K. and Minton, N.P. (1990) Physical characterisation of the replication region of the

è dicale and the University of Paris V. Me

L

Streptococcus faecalis

plasmid

pAM 1. Gene 87, 79^90. [17] Rose, R.E. (1988) The nucleotide sequence of pACYC184. Nucleic Acids Res. 16, 355.

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